The present invention relates to novel immortalized middle ear epithelial cell lines and their use in screening assays.
Otitis media (OM), or inflammation of the middle ear, is the second most frequent illness resulting in visits to physicians following the common cold and the most common cause of hearing impairment in children. According to the Centers for Disease Control and Prevention/National Center for Health Statistics, OM accounts for an estimated 31 million annual visits to the doctor""s office. Eighty percent of the children born each year experience at least one episode of OM by their third birthday, and one in three have repeated bouts of the disease. Although the fraction of health care expenditure taken up by OM is unknown, it is estimated to have a yearly cost exceeding $5 billion.
Acute OM (AOM) generally refers to the rapid onset of signs and symptoms of an acute infection in the middle ear. Chronic otitis media with effusion (OME), also known as persistent middle ear effusion, is the major sequela of acute OM. OME is characterized by the accumulation of serous, mucoid or purulent fluid in the middle ear space, without compromising the intactness of the tympanic membrane (Bluestone, C. and Klein, J O. 1995. Otitis media in infants and children: W.B. Saunders Company). In approximately 40% of the cases, middle ear effusion can still be seen one month after antibiotic treatment and in 20% of them, even after three months. In the US alone, about one million tympanostomy tubes are inserted per year, for the treatment of persistent or recurring OM. The course of OME is generally benign and self-limiting. The deafness caused by the effusions, however, if not treated in time, could adversely affect the child""s development and educational progress.
OM is caused mainly by three pathogens: Streptococcus pneumoniae, Moraxella catarrhalis and non-typeable Haemophilus influenzae (NTHI) [Block, S. L. 1997. Causative pathogens, antibiotic resistance and therapeutic considerations in acute otitis media. Pediatr Infect Dis J. 16: 449-56; Brook, I. 1994. Otitis media: microbiology and management. J Otolaryngol. 23: 269-75; Maxson, S., T. Yamauchi. 1996. Acute otitis media. Pediatr Rev. 17: 191-5; and Strausbaugh, L. 1997. Haemophilus influenzae infections in adults: a pathogen in search of respect. Postgrad Med. 101:191-200]. The inflammatory reaction caused by the interaction of bacterial surface components with the epithelial cells of the middle ear is one of the most crucial steps in the development of otitis media. Many of the processes involved however, remain poorly understood [DeMaria, T. F., Yamaguchi, T., Bakaletz, L. O., and Lim, D. J. (1992). Serum and middle ear antibody response in the chinchilla during otitis media with effusion induced by nonviable nontypeable Haemophilus influenzae. J Infect Dis 165, S196-.; Ernst, (1999). Review article: the role of inflammation in the pathogenesis of gastric cancer. Aliment Pharmacol Ther 13, 13-18; Giebink, G. S. (1999). Otitis media: the chinchilla model. Microb Drug Resist 5, 57-72; Patel, J., Faden, H., Sharma, S., and Ogra, P. L. (1992). Effect of respiratory syncytial virus on adherence, colonization and immunity of non-typable Haemophilus influenzae: implications for otitis media. Int J Pediatr Otorhinolaryngol 23, 15-23; and Weinberg, A., Krisanaprakornkit, S., and Dale, B. A. (1998) Epithelial antimicrobial peptides: review and significance for oral applications. Crit Rev Oral Biol Med 9, 399-414]. In order elucidate the cellular and molecular mechanisms involved in the pathogenesis of otitis media, it is important to understand the biology of the middle ear epithelium and such studies have traditionally required large numbers of middle ear epithelial cells. Although there have been several reports on the establishment of primary cultures and cell lines of middle ear epithelial cells in rodents, to date there have been no reports of immortalized human middle ear epithelial cells. Thus, there is a need for cell lines derived from normal human middle ear that can be used, to study the molecular mechanisms involved in the pathogenesis of otitis media.
Primary cultures of untransformed human cells are generally difficult to propagate for extended periods. Their life span in culture is very limited and they usually become senescent after 4-5 passages. Although middle ear and Eustachian tube epithelial cell lines have been derived from laboratory animals [Portier F., et al. Oxygen modulates Na+ absorption in middle ear epithelium. Am J Physiol 1999 Feb;276(2 Pt 1):C312-7; van Blitterswijk C A, et al. Culture and characterization of rat middle-ear epithelium. Acta Otolaryngol (Stockh). 1986 May-Jun; 101 (5-6):453-66; Takeno S, et al. Tissue culture of middle ear epithelium using fibroblast-reorganized collagen gels. J Otolaryngol. 1993 Oct;22(5):3804; de Serres L M, et al. Bioelectric properties of gerbil middle ear epithelia. Arch Otolaryngol Head Neck Surg 1991 Apr;117(4):416-21; Herman P, et al. Ion transport by primary cultures of Mongolian gerbil middle ear epithelium. Am J Physiol 1992 Mar;262(3 Pt 2):F373-80; Ueyama S., et al. Immortalization of rat middle ear epithelial cells by Adenol2-SV40 hybrid. Ann Otol Rhinol Laryngol. (in print); Herman P., et al. Middle ear cell line that maintains vectorial electrolyte transport. J Cell Physiol. 1993 Mar;154(3):615-22.; and Nakamura A, et al. Serial culture and characterization of the chinchilla middle era epithelium. Ann Otol Rhinol Laryngol. 1991 Dec;100(12): 1024-31.], to date there have been no report of immortailized human middle ear epithelial cells.
Expression of viral genes is an effective way of immortalizing primary cells. Among the most commonly used and effective transforming viral sequences are the E6 and E7 genes of the human papilloma virus (HPV) type 16, which function to dysregulate cell growth. HPV is a site specific DNA virus that is known to infect the basal cell layers and replicate during epithelial cell differentiation [Woodworth CD, et al. Characterization of normal human exocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18 DNA. Cancer Res. 1988 Aug 15;48(16):4620-8; Pecoraro G, et al. Differential effects of human papilloma virus type 6, 16, and 18 DNAs on immortalization and transformation. Proc Natl Acad Sci USA. 1989 Jan; 86(2): 563-7; Band V, et al. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements. Proc Natl Acad Sci U S A. 1990 Jan;87(1):463-7; Durst M, et al. Inverse relationship between human papillomavirus type 16 early gene expression and cell differentiation in nude mouse epithelial cysts and tumors induced by HPV-positive human cell lines. J Virol 1991 Feb;65(2):796-804; Merrick DT, et al. Altered expression of proliferation and differentiation markers in human papillomavirus 16 and 18 immortalized epithelial cells grown in organotypic culture. Am J Pathol. 1992 Jan;140(1):167-77; Pirisi L, et al. Continuous cell lines with altered growth and differentiation properties originate after transfection of human keratinocytes with human papillomavirus type 16 DNA. Carcinogenesis. 1988 Sep;9(9):1573-9; Willey J C, et al. Immortalization of normal human bronchial epithelial cells by human papillomaviruses 16 or 18. Cancer Res. 1991 Oct 1;51(19):5370-7; Woodworth C D, et al. Human cervical and foreskin epithelial cells immortalized by human papillomavirus DNAs exhibit dysplastic differentiation in vivo. Cancer Res 1990 Jun 15;50(12):3709-15; and Blanton R A, Perez-Reyes N, Merrick D T, McDougall J K. Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture. Am J Pathol. 1991 Mar;138(3):673-85]. More than 80 different types of human papilloma viruses (HPVS) have now been isolated from a variety of squamous epithelial lesions, and approximately 18 of them have been associated with anogenital tract lesions. Some of these, such as HPV type 6 (HPV-6) and HPV-11, are generally associated with benign proliferative lesions, including condyloma acuminata, which only infrequently progress to cancers. Others, such as HPV-16, HPV-18, HPV-31, HPV-33, and HPV-35, are associated with genital tract lesions, which are at risk for malignant progression, and with genital tract cancers [Ostrow R S, et al. A survey of human cancers for human papillomavirus DNA by filter hybridization. Cancer. 1987 Feb 1;59(3):429-34; and Turek L P, et al. The genetic program of genital human papillomaviruses in infection and cancer. Obstet Gynecol Clin North Am 1996 Dec;23(4):735-58].
Introduction of HPV 16 DNA into cells results in the immortalization of the cells at a high frequency and is independent of the genetic characteristics of the host cells [Woodworth C D, et al. (1988); Pecoraro G, et al.; Band, V., et al.; Durst M, et al.; Merrick D T, et al.; Pirisi L, et al.; Willey J C, et al.; Woodworth C D, et al. (1990); and Blanton R A, et al.]. Immortalization of human cells by HPV DNA is usually associated with aneuploidy and rearrangement of chromosomes [Oda D, et al. Chromosomal abnormalities in HPV-16-immortalized oral epithelial cells. Carcinogenesis 1996 Sep;17(9):2003-8; Hietanen S, et al. Isolation of two keratinocyte cell lines derived from HPV-positive dysplastic vaginal lesions. Int J Cancer 1992 Sep 30;52(3):391-8; Hashida T, et al. Induction of chromosome abnormalities in mouse and human epidermal keratinocytes by the human papillomavirus type 16 E7 oncogene. J Gen Virol 1991 Jul;72(Pt 7):1569-77; Smith P P, et al. Cytogenetic analysis of eight human papillomavirus immortalized human keratinocyte cell lines. Int J Cancer 1989 Dec 15;44(6):1124-31; and Klemi P J, et al. Association of DNA aneuploidy with human papillomavirus-induced malignant transformation of sinonasal transitional papillomas. Otolaryngol Head Neck Surg 1989 Jun;100(6):563-7]. Only those cells with integrated copies of HPV DNA become permanent lines, suggesting that genetic alterations caused by viral DNA integration and expression are necessary for continuous growth. HPV-16 or HPV-18 DNA has been found integrated in a high percentage of cervical carcinomas and in cell lines derived from these cancers. This is in contrast with the premalignant dysplastic lesions associated with HPV-16 and HPV-18, in which the viral DNA is usually found in an extrachromosomal state.
In several cases in which the number of integrated viral genomes was low enough to permit a detailed analysis, the integration pattern revealed remarkable specificity with respect to the circular viral genome [Yokoyama M, et al. Alterations in physical state and expression of human papillomavirus type 18 DNA following crisis and establishment of immortalized ectocervical cells. Virus Res 1995 Jul;37(2):139-51; Gilles C, et al. Viral integration sites in human papilloma virus-33-immortalized cervical keratinocyte cell lines. Cancer Genet Cytogenet 1996 Aug;90(1):63-9; Fontijn R, et al. Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. Exp Cell Res 1995 Jan;216(1):199-207; and DiPaolo J A, et al. Cellular and molecular alterations in human epithelial cells transformed by recombinant human papillomavirus DNA. Crit Rev Oncog 1993;4(4):337-60]. Integration occurs in the E1-E2 region, disrupting the E2 viral transcriptional regulatory circuitry. The E2 open reading frame (ORF), as originally demonstrated with the bovine papillomavirus type 1, encodes both positive- and negative-acting transcriptional regulatory factors. For HPV-16 and HPV-18, E2 appears to act principally as a repressor of the promoter from which the E6 and E7 genes are transcribed [Demeret C, et al. Different mechanisms contribute to the E2-mediated transcriptional repression of human papillomavirus type 18 viral oncogenes. J Virol 1997 Dec;71(12):9343-9; Rapp B, et al. Cell-type-specific separate regulation of the E6 and E7 promoters of human papillomavirus type 6a by the viral transcription factor E2. J Virol 1997 Sep;71(9):6956-66; Dowhanick J J, et al. Suppression of cellular proliferation by the papillomavirus E2 protein. J Virol 1995 Dec;69(12):7791-9; Thierry F, et al. Functional analysis of E2-mediated repression of the HPV18 P105 promoter. New Biol 1991 Jan;3(1):90-100.; Bernard B A, et al. The human papillomavirus type 18 (HPV18) E2 gene product is a repressor of the HPV18 regulatory region in human keratinocytes. J Virol 1989 Oct;63(10):4317-24; Goodwin E C, et al. Transactivation-competent bovine papillomavirus E2 protein is specifically required for efficient repression of human papillomavirus oncogene expression and for acute growth inhibition of cervical carcinoma cell lines. J Virol 1998 May;72(5):3925-34 and Ruley H E. Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature 1983 Aug 18-24;304(5927):602-6]. The HPV genomes in cervical carcinomas and in derived cell lines are transcriptionally active, and the patterns of viral mRNA species are specific, with regular expression of the E6 and E7 ORFs.
The E7 ORF of HPV-16 encodes a 21-kilodalton phosphoprotein, and the E7 genes of HPV-16 and HPV-18 are sufficient for focus formation of established rodent fibroblasts such as NIH 3T3 cells. The E7 protein is functionally and structurally related to the adenovirus E1A proteins (AdE1A); it can transactivate the AdE2 promoter and can cooperate with an activated ras oncogene to transform primary rat cells. The amino-terminal 38 amino acids of E7 are strikingly similar to portions of conserved domain 1 (amino acids 37 to 49) and domain 2 (amino acids 116 to 137) of the AdE1A proteins as well as to portions of the large tumor antigens (T) of papovaviruses. The AdE1A, simian virus 40 (SV40) T, and HPV-16 E7 proteins form specific complexes with the product of the retinoblastoma tumor suppressor gene (p105-RB), and complex formation with p105-RB is mediated through these conserved sequences for AdE1A and SV40 T as well as for HPV-16 E7.
The transforming potential of the E6 gene has been less well defined [Villa L L, et al. Differences in transformation activity between HPV-18 and HPV-16 map to the viral LCR-E6-E7 region. Virology 1991 Mar;181(1): 374-7; Wilson S E, et al. Expression of E6/E7 or SV40 large T antigen-coding oncogenes in human corneal endothelial cells indicates regulated high-proliferative capacity. Invest Ophthalmol Vis Sci 1995 Jan;36(1):32-40; Halbert C L, et al. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J Virol 1991 Jan;65(1):473-8; and Barbosa M S, et al. In vitro biological activities of the E6 and E7 genes vary among human papillomaviruses of different oncogenic potential. J Virol. 1991 Jan;65(1):292-8]. In NIH 3T3 fibroblasts, it may contribute to characteristics of the transformed phenotype such as anchorage independence or tumorigenicity in nude mice. In human cells, E6 appears to cooperate with the E7 oncoprotein in mediating-cellular immortalization. Recently, it has been demonstrated that E6 binds to, and mediates the degradation of, the cellular tumor suppressor protein p53 [Band V, et al. Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells. J Virol. 1991 Dec;65(12):6671-6].
It has been shown recently that both the E6 and E7 ORFs are necessary for the extension of the life span of human diploid fibroblasts. Mutation studies of the early HPV-16 genes that directly participate in the in vitro transformation of primary human keratinocytes have shown that both the full-length E6 and E7 genes are required for induction of keratinocyte immortalization and resistance to terminal differentiation. Keratinocyte transformation with HPV-18 DNA requires only the HPV-18 regulatory region and the E6/E7 genes which induce two progressive steps in cellular transformation [Hudson J B, et al. Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18. J Virol 1990 Feb;64(2):519-2; Munger K, et al. The E6 and E7 genes of the human papillomavirus Type 16 together are necessary and sufficient for transformation of primary human keratinocytes, vol. 63, No. 10, Journal of Virology, Oct. 1989, pp. 4417-4421; and Barbosa M S, et al. The E6 and E7 genes of HPV-18 are sufficient for inducing two-stage in vitro transformation of human keratinocytes,. Oncogene 1989 Dec;4(12):1529-32].
There is a need for cell lines derived from normal human middle ear epithelium which can be used, in turn, to study the molecular mechanisms involved in the pathogenesis that results in hearing loss. Furthermore, once derived, such cell lines can be used for a variety of purposes, including drug discovery, toxicity assays, discovery of novel genes important for middle ear epithelial cell function, tissue engineering and bionic device development.
The present invention is directed to a method of immortalizing middle ear epithelial cells that satisfies the need for cell lines derived from normal human middle ear epithelium, which can be used for in vitro assays to determine the molecular mechanisms involved in the pathogenesis of hearing disorders, including otitis media.
One version of the invention is a method for producing a non-tumorigenic immortalized cell line that retains phenotypic properties of middle ear epithelial cells. The first step of the method is providing a primary cell culture of human middle ear epithelial cells. The next step is introducing a polynucleotide encoding an exogenous immortalizing gene into the middle ear epithelial cells. The last step is selecting for immortalized cells that express the exogeous immortalizing gene and retain phenotypic properties of middle ear epithelial cells. The polynucleotide is typically a subgenomic fragment of a virus, such as SV40, adenovirus, and human papilloma virus. Preferably, the immortalizing gene is the E6 and E7 genes of said human papilloma virus types 16, 18, 31, 33, or 35, with type 16 being most preferred. The polynucleotide is typically contained in a viral or plasmid vector, such as a retrovirus, adenovirus, or adeno-associated virus vector, most preferably a replication-defective retrovirus construct.
Another version of the invention is a substantially pure cell line of immortalized non-tumorigenic human middle ear epithelial cells, which expresses an exogenous immortalizing gene, such as SV40 T antigen, adenovirus E1A, or human papilloma virus E6 and E7 genes. Preferably the immortalizing gene is the E6 and E7 genes of human papilloma virus types 16, 18, 31, 33, and 35, with type 16 being most preferred.
In another version of the present invention, the substantially pure cell line of immortalized human middle ear epithelial cells actively expresses the E6 and E7 gene of human papilloma virus 16, wherein the immortalized cell line maintains phenotypic characteristics of human middle ear epithelial cells, such as immunostaining positive of for cytokeratins 4, 7, and 18, but not desmin, vimentin, and Factor VIII. The cells do not have a neoplastic phenotype and exhibit contact inhibition and anchorage dependence.
A most preferred version of the present invention is a cell line having the identifying characteristics of ATCC Accession # CRL PTA-81.
Another version of the present invention is a method for determining the effect of a pharmacological agent on cells of the middle ear. The first step of the method is contacting the immortalized middle ear epithelial cell line with a pharmacological agent and then determining the effect of the pharmacological agent on the cell line. The effect can be a change in cell growth, a change in a phenotypic characteristic of the cell line, or an increase or decrease in expression of a cellular gene. The effected cellular genes can be mucins, cytokines, growth factors and molecules of innate immunity, including, but not limited to, defensins, surfactant proteins, lysozyme, and lactoferrin. Moreover, the pharmacological agent can belong to a family such as chemicals, drugs, hormones, cytokines, and growth factors.
The present invention also provides a kit for screening a pharmacological agent on middle ear epithelial cells, which includes a container of the immortalized middle ear epithelial cells described above.